EP0531436A1 - Agents de reticulation non photoactives, specifiques a une sequence, qui se lient au sillon principal d'adn duplex - Google Patents

Agents de reticulation non photoactives, specifiques a une sequence, qui se lient au sillon principal d'adn duplex

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Publication number
EP0531436A1
EP0531436A1 EP91911335A EP91911335A EP0531436A1 EP 0531436 A1 EP0531436 A1 EP 0531436A1 EP 91911335 A EP91911335 A EP 91911335A EP 91911335 A EP91911335 A EP 91911335A EP 0531436 A1 EP0531436 A1 EP 0531436A1
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European Patent Office
Prior art keywords
duplex
sequence
agent
crosslinking agent
crosslinking
Prior art date
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EP91911335A
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German (de)
English (en)
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EP0531436A4 (en
Inventor
Mark D. Matteucci
Steven Krawczyk
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Gilead Sciences Inc
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Gilead Sciences Inc
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Publication of EP0531436A1 publication Critical patent/EP0531436A1/fr
Publication of EP0531436A4 publication Critical patent/EP0531436A4/en
Withdrawn legal-status Critical Current

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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma

Definitions

  • the invention relates generally to compositions useful in "antisense” therapy and diagnosis. More particularly, it concerns compositions which are capable of binding in a sequence-specific manner to the major groove of nucleic acid duplexes and forming covalent bonds with one or both strands of the duplex.
  • Antisense therapies are generally understood to be those which target specific nucleotide sequences associated with a disease or other undesirable condition. While the term “antisense” appears superficially to refer specifically to the well-known A-T and G-C complementarity responsible for hybridization of a "sense" strand of DNA, for example, to its “antisense” strand, this term, as applied to the technology, has come to be understood to include any mechanism for interfering with those aspects of the disease or condition which are mediated by nucleic acids.
  • compositions and methods useful in the invention target the major groove of nucleic acid duplexes in sequence dependent manner. In order to distinguish targeted duplexes from those which are indigenous to the subject or which otherwise are not desired to be affected, this binding must be sequence specific. It is now known that single-stranded oligonucleotides are capable of sequence-specific binding to the major groove in a duplex according to rules which have been reported, for example, by Moser and Dervan, Science (1987) 238:645-650. In this report, sequence- specific recognition was used to associate homopyrimidine derivatized EDTA with the major groove and effect cleavage of the double helix.
  • Vlassov, V.V., et al., Nucleic Acids Res (1986) 14:4065- 4076 describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences.
  • a report of similar work by the same group is that by Knorre, D.G., et al.. Biochimie (1985) .67:785-789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (J Am Chem Soc (1987) 109:1241-1243) . Meyer, R.B., et al., J Am Chem Soc
  • Vlassov, V.V. et al. Gene (1988) 313-322 and Fedorova, O.S. et al., FEBS (1988) 228:273- 276, describe targeting duplex DNA with a 5'-phospho- linked oligonucleotide.
  • the invention provides crosslinking agents which associate in a sequence-specific manner to the major groove of nucleic acid duplexes to obtain triple helical products which are stabilized by covalent bonds.
  • the stabilized triplex may be optionally subjected to conditions which result in cleavage of the duplex.
  • the stabilized binding of the sequence-specific crosslinking agent permits either interruption of the normal function of the duplex (for example, in replication) or, in the case of regulatable duplexes (for example, associated with transcription) , may enhance the activity of the target duplex.
  • the resulting triple- helical complex may become more or less susceptible to cleavage under ambient or .in situ conditions. Stimulation of cleavage may be desirable in the case of therapeutic regimens designed to inactivate the target DNA; it is also useful in diagnostic assays by permitting facile detection of covalently bound probes.
  • the invention is directed to crosslinking agents which associate with the major groove of nucleic acid duplexes in a sequence-specific manner and which effect at least one covalent crosslink to at least one strand of the duplex. Multiple crosslinks may also be formed, with one or both of the duplex strands, depending on the design of the crosslinking agent.
  • Preferred crosslinking agents are oligonucleotides, which take advantage of the duplex sequence-coupling rules known in the art, and peptide sequences, which can be designed to mimic regulatory peptides which recognize specific sequences.
  • the moiety which performs the crosslinking function of the crosslinking agent results in the formation of covalent bonds in a pattern dependent on the design of the agent.
  • the invention is directed to methods to form triple helical complexes containing sequence-specific agents covalently bound in the major groove, which method comprises contacting the target duplex with a crosslinking reagent of the invention.
  • the invention is directed to the resulting triple helical complexes, and to methods for therapy and diagnosis using the crosslinking reagents of the invention.
  • Figure 1 shows the structures of preferred alkylating agents which effect the crosslinking of the sequence-specific agents of the invention.
  • Figure 2 outlines the procedure for preparation of the N ,N -ethanocytosine-containing oligomers that are preferred crosslinking reagents of the invention.
  • Figure 3 shows the construction of a tetracassette duplex designed to assess the specificity of the reagents of the invention.
  • Figure 4 shows the results of an assay showing the sequence specificity of the invention crosslinking agent.
  • Figure 5 shows the results of treatment of target sequences with the reagents of the invention with and without cleavage of the complexes.
  • the invention provides reagents which are capable of sequence-specific binding in the major groove of a nucleic acid duplex and which are also capable of forming covalently bonded crosslinks with the strands of the duplex without the necessity for photoactivation.
  • moieties to effect the covalent bonding are employed which do not override the sequence specificity of the remainder of the reagent.
  • the moiety which effects the covalently bonded crosslink is itself specific for a particular target site in a preferred embodiment.
  • Sequence specificity is essential to the utility of the reagents of the invention. If not capable of distinguishing characteristic regions of a target from those of duplexes which are not to be targeted, the reagents would not behave in a manner compatible with their function as either therapeutic or diagnostic agents. Accordingly, it is essential that despite the reactivity of the moiety which effects covalent binding, this activity not be so kinetically favored that sequence specificity is lost.
  • Sequence specificity can be conferred in a manner consistent with the chemical nature of the reagent.
  • the specificity is conferred by providing a region of spatial and charge distribution which allows close association between the reagent and the charge and spatial contours of the major groove of the target duplex.
  • This association and sequence specificity are defined in terms of the ability of the reagent to distinguish between target sequences in a sample which differ in one or more basepairs.
  • the reagents of the invention can discriminate between regions of duplexes which differ by as few as 1 basepair out of 5, preferably l basepair out of 10, more preferably 1 basepair out of 15, and most preferably 1 basepair out of 20, in in vivo or in vitro culture conditions or under conditions of the diagnostic assay.
  • the stringency of the criterion varies with the length of the region, since larger regions can tolerate more mismatches than smaller ones under the same conditions.
  • a highly discriminatory reagent could detect a mismatch of only 1 basepair in a sequence of 20 basepairs; a more sequence-specific reagent could detect this 1-basepair difference in a region of 30 basepairs.
  • the reagents of the invention are capable of at least discriminating between differences of 1 basepair in a 5- mer target, preferably 1 basepair in a 10-mer target, and most preferably 1 basepair in a 20-mer target.
  • association of the oligonucleotide sequence specificity-conferring region of the reagent can be manipulated by utilizing either or both CT or GT binding to one or both strands of the target duplex.
  • "switchback" oligomers are described which contain one or more regions of inverted polarity.
  • One application of such "switchback" oligomers includes the ability to design reagents which cross over between the two strands of the duplex using parallel association with the purine regions of the strands of the duplex. Alternatively, this crossover could be effected by modifying the oligonucleotide sequence to switch back between parallel and antiparallel modes of association with the major groove.
  • sequence specificity can be designed relative to either or both strands of the duplex.
  • Oligonucleotide is understood to include both DNA and RNA sequences and any other type of polynucleotide which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base.
  • the term "nucleoside” or “nucleotide” will similarly be generic to ribonucleosides or ribonucleotides, deoxyribonucleosides or deoxyribonucleotides, or to any other nucleoside which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base.
  • nucleoside and nucleotide include those moieties which contain not only the known purine and pyrimidine bases, but also heterocyclic bases which have been modified. Such modifications include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles.
  • Nucleosides or nucleotides also include those which contain modification in the sugar moiety, for example, wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or functionalized as ethers, amines, and the like. Examples of modified nucleosides or nucleotides include, but are not limited to:
  • 5-methyluridine (2'-deoxy-5-methyluridine is the same as thymidine) inosine 2 -deoxy-inosine xanthosine 2 deoxy-xanthosine
  • one or more nucleotides may contain this linkage.
  • Oligonucleotides may contain conventional internucleotide phosphodiester linkages or may contain modified forms such as phosphoramidate linkages. These alternative liking groups include, but are not limited to embodiments wherein a moiety of the formula P(0)S, P(0)NR 2 ,, P(0)R, P(0)0R', CO, or CNR 2 , wherein R is H (or a salt)or alkyl (1-6C) and R' is alkyl (1-6C) is joined to adjacent nucleotides through -O- or -S-. Not all such linkages in the same oligomer need to be identical. Inversions of polarity can also occur in "derivatives" of oligonucleotides.
  • oligonucleotides may be covalently linked to various moieties such as intercalators, substances which interact specifically with the minor groove of the DNA double helix and other arbitrarily chosen conjugates, such as labels (radioactive, fluorescent, enzyme, etc.). These additional moieties may be derivatized through any convenient linkage.
  • intercalators such as acridine can be linked through any available -OH or -SH, e.g., at the terminal 5' position of RNA or DNA, the 2 1 positions of RNA, or an OH or SH engineered into the 5 position of pyrimidines, e.g., instead of the 5 methyl of cytosine, a derivatized from which contains -CH 2 CH 2 CH 2 OH or -CH 2 CH 2 CH 2 SH in the 5 position.
  • substituents can be attached, including those bound through conventional linkages.
  • the -OH moieties in the oligomers may be replaced by phosphonate groups, protected by standard protecting groups, or activated to prepare additional linkages to other nucleotides, or may be bound to the conjugated substituent.
  • the 5 1 terminal OH may be phosphorylated; the 2'-OH or OH substituents at the 3' terminus may also be phosphorylated.
  • the hydroxyls may also be derivatized to standard protecting groups.
  • a suitably protected nucleotide having a cyanoethylphosphoramidite at the position to be coupled is reacted with the free hydroxyl of a growing nucleotide chain derivatized to a solid support.
  • the reaction yields a cyanoethylphosphonate, which linkage must be oxidized to the cyanoethylphosphate at each intermediate step, since the reduced form is unstable to acid.
  • the phosphonate-based synthesis is conducted by the reaction of a suitable protected nucleoside containing a phosphonate moiety at a position to be coupled with a solid phase-derivatized nucleotide chain having a free hydroxyl group, in the presence of a suitable catalyst to obtain a phosphonate linkage, which is stable to acid.
  • a suitable catalyst to obtain a phosphonate linkage, which is stable to acid.
  • the oxidation to the phosphate or thiophosphate can be conducted at any point during the synthesis of the oligonucleotide or after synthesis of the oligonucleotide is complete.
  • the phosphonates can also be converted to phosphoramidate derivatives by reaction with a primary or secondary amine in the presence of carbon tetrachloride.
  • Non-phosphorous based linkages may also be used, such as the formacetyl type linkages described and claimed in co-pending applications U.S. Serial Nos. 426,626 and 448,914, filed on 24 October 1989 and 11 December 1989, both assigned to the same assignee and both incorporated herein by reference.
  • oligonucleotides may also be synthesized using solution phase methods such as triester synthesis. These methods are workable, but in general, less efficient for oligonucleotides of any substantial length.
  • solution phase methods such as triester synthesis.
  • the ability of the candidate crosslinking reagent to effect covalent bonding to the target duplex can be assessed in simple assays using either a shift in electrophoresis gel mobility or assessment of size after cleavage.
  • the template can be advantageously labeled at a terminus using, for example, ⁇ -P32 dATP and Klenow.
  • the labeled template and the candidate oligonucleotide are then incubated under suitable conditions to effect triplex binding.
  • For the shift assay they are then analyzed on a 6% denatured polyacrylamide gel after addition of an equal volume of formamide denaturant.
  • the shift in mobility verifies binding to form the triplex and resistance to denaturation.
  • the covalent crosslinking moiety associated with the reagent is also capable of effecting cleavage of the duplex under appropriate conditions, the location of binding by the reagent can readily be ascertained by application of the sample to size separation techniques. Multiple binding to more than one cassette will result in multiple small fragments; binding to only one of the cassettes results in a single defined fragment of the labeled DNA of predicted size. Thus, even without prior knowledge of design rules for specific association, candidate reagents can conveniently be tested with suitably labeled cassette-containing DNA.
  • the crosslinking agent is a moiety which is capable of effecting at least one covalent bond between the crosslinking agent and the duplex. Multiple covalent bonds can also be formed by providing a multiplicity of such moieties.
  • the covalent bond is preferably to a base residue in the target strand, but can also be made with other portions of the target, including the saccharide or phosphodiester.
  • the reaction nature of the moiety which effects crosslinking determines the nature of the target in the duplex.
  • Preferred crosslinking moieties include acylating and alkylating agents, and, in particular, those positioned relative to the sequence specificity-conferring portion so as to permit reaction with the target location in the strand.
  • the crosslinking moiety can conveniently be placed as an analogous pyrimidine or purine residue in the sequence.
  • the placement can be at the 5 1 and/or 3' ends, the internal portions of the sequence, or combinations of the above. Placement at the termini to permit enhanced flexibility is preferred.
  • Analogous moieties can also be attached to peptide backbones.
  • a switchback oligonucleotide containing crosslinking moieties at either end can be used to bridge the strands of the duplex with at least two covalent bonds.
  • nucleotide sequences of inverted polarity can be arranged in tandem with a multiplicity of crosslinking moieties to strengthen the complex.
  • alkylating moieties that are useful in the invention are those shown in Figure 1. These are derivatized purine and pyrimidine bases which can be included in reagents which are oligomers of nucleotides as described above. As seen in Figure 1, heterocyclic base analogs which provide alkyl moieties attached to leaving groups or as aziridenyl moieties are shown. (“Aziridenyl” refers to an ethanoamine substituent of the formula ⁇ N /.)
  • the agent can also contain additional components which provide additional functions.
  • additional components which provide additional functions.
  • ligands which effect transport across cell membranes, specific targeting of particular cells, stabilization of the triplex by intercalation, or moieties which provide means for detecting the oligomer alone or in the context of the triple helix formed can be included.
  • the crosslinking agents of the invention may thus be further conjugated to lipid-soluble components, carrier particles, radioactive or fluorescent labels, specific targeting agents such as antibodies, and membrane penetrating agents and the like.
  • the specific crosslinking agents of the invention are useful in therapy and diagnosis.
  • the agents are designed to target duplexes for either interruption or enhancement of their function.
  • suitable target genes for enhanced function include those which control the expression of tumor suppressor genes (Sager, Science (1989) 246:1406) or for duplexes which control the expression of cytokines such as IL-2.
  • cytokines such as IL-2.
  • complexing into the major groove may result in blocking the function of the target duplex as would be desirable where the duplex mediates the progress of a disease, such as human immunodeficiency virus, hepatitis-B, respiratory syncytial virus, herpes simplex virus, cytomegalovirus, rhinovirus and influenza virus.
  • other undesirable duplexes are formed in various malignancies, including leukemias, lung, breast and colon cancers, and in other metabolic disorders.
  • crosslinking agents of the invention depends, of course, on their chemical nature, and on the nature of the condition being treated. Suitable formulations are available to those of ordinary skill, and can be found, for example, in Remington's Pharmaceutical Sciences. latest edition. Mack Publishing Co., Easton, PA. Dosage levels are also determined by the parameters of the particular situation, and as is ordinarily required in therapeutic protocols, optimization of dosage levels and modes of administration are within ordinary and routine experimentation.
  • X represents N N -ethanocytosine deoxynucleotide are synthesized as outlined in Figure 2.
  • the steps in the synthesis refer to Webb and Matteucci, Nucleic Acids Res (1986) 14.:5399-5467 and Froehler and Matteucci,
  • test cassettes contain identical sequences except for a single base.
  • Az-A is designed to associate specifically with cassette 1; Az-B is designed to- associate specifically with cassette 2.
  • This target DNA is an end-labeled PvuII-Sal fragment containing these cassettes separated by convenient restriction sites. The N 4N4 cytosine moiety was expected to crosslink covalently only to a guanine residue.
  • the target plasmid was supplied in 1 ⁇ l volume at 50,000 cpm/ ⁇ l, Az-A and Az-B were supplied in 1 ⁇ l aliquots of 500 ⁇ M concentration and the volume was made up in all reaction mixtures to 10 ⁇ l with water.
  • the mixtures were incubated for 13.5 hr at room temperature (23-25°C) .
  • Lane 1 represents reaction mix 1 to which DMS was added. Extensive degradation is seen.
  • Lane 2 is the reaction mixture which contained Az-A. As shown, treatment with pyrrolidine yields mainly one degradation product, the size of which corresponds to the labeled fragment that would be obtained if cleavage occurred in cassette 1.
  • Lane 3 shows the results from reaction mix 3 containing Az-B. Again, a single prominent degradation fragment was obtained which corresponds in size to the labeled fragment which would be obtained if cleavage occurred in cassette 2.
  • the pyrrolidine control in lane 4 shows only modest random degradation.
  • oligonucleotides 2-6 include the base analogs aziridinylcytosine (N4,N4-ethanocytosine) , designated "Z" in the tabulated sequences and 5-methylcytosine, designated C in the table.
  • X indicates 1,3-propanediol.
  • the 5-methyl-C groups were FMOC-protected and an oxalyl-CPG support (R. Letsinger, personal communication, described below) was used for the synthesis.
  • the base representing the 5 ' terminus was coupled to a CPG support for the production of the 0DN s using the following method (R. Letsinger, personal communication) .
  • Oxalyl chloride (20 ⁇ l, 0.23 mmol) was added to a solution of 1,2,4-triazole (77 mg, 1.1 mmol) in acetonitrile (2 ml) .
  • the support bound H-phosphonate oligomer was oxidized with I 2 /pyridine/H 2 0 tw: ⁇ ce for 30 m:Ln and subsequently converted to the free oligonucleotide by deprotection and cleavage from the support by treatment with 20% aziridine in DMF for 2 hours at room temperature.
  • the oligomers were recovered and further purified by running the reaction mixture from the synthesis machine over NAP-5 (Pharmacia Sephadex G-25) column to remove salts, free aziridinylcytosine residues, FMOC blockers, etc.
  • NAP-5 column was used according to the manufacturers directions.
  • the potentially covalent binding moiety, Z is at the 3 1 terminus of the oligomer in ODN3, at the 3 1 end in ODN4, at both ends in ODN5 and internal to the oligomer in ODN6.
  • lane 1 represents the untreated duplex target, and shows no difference from lane 2 which was treated with 0DN2, containing no crosslinking moiety.
  • Lanes 3 and 4 represent the results of reaction mixtures using ODNs 3 and 4 respectively; in both cases, considerable reaction has occurred; this reaction is virtually complete in lane 5 which represents treatment with 0DN5.
  • Lane 6 indicates that although some reaction occurred with 0DN6, this was less effective when the covalent binding moiety is internal to the oligomer.
  • Example 4 Additiional Crosslinking Agents
  • the modified nucleoside N-methyl-8-oxo-2'-deoxyadenine (MODA) is designated “M”
  • 5-methylcytosine is represented by “C”
  • nucleosides containing an aziridenyl group N 4N4- ethanocytosine
  • Z 1 nucleosides containing an aziridenyl group
  • some of the oligomers contain an inverted polarity region, in this illustration formed from an o-xyloso di er synthon.
  • the linking group, o- xyloso (nucleotides that have xylose sugar linked via the o-xylene ring), is designated “X”.
  • Crosslinking agents that bind to certain HIV targets are as follows. For binding to the 5'- GGAAAAGGAAGGAAATTTC-3* sequence:
  • the oligonucleotides are labeled by kinasing at the 5' end and are tested for their ability to bind target sequence under conditions of 1 mM spermine, 1 mM MgCl 2 , 140 mM KCl, 10 mM NaCl, 20 mM MOPS, pH 7.2 with a target duplex concentration of 10 pM at 37°C for 1 hour. These conditions approximate physiological conditions, and the binding is tested either in a footprint assay, or in a gel-shift assay essentially as described in Cooney, M. et al., Science (1988) 241:456-459.
  • HUMIL1B Human Interleukin-1 Beta Gene
  • illustrative nucleotides are : a. for HUMIL1B beginning at neucleotide 6379 104 5•-ZTTTTMTTMTM-X 1 -TMTTTT-5• , b. for HUMIL1B beginning at neucleotide 7378
  • HUMTNFAA Human Tumor Necrosis Factor
  • the illustrative nucleotides are: a. for HUMTNFAA beginning at neucleotide 251 203 5'-TMTMMMTTM-X 3 -MMMMZ-5• , b. for HUMTNFAA beginning at neucleotide 1137
  • illustrative nucleotides are: a. for HUMINT02 beginning at neucleotide 1612 302 5 '-TCTTMCTT-X 4 -MTTCTMZ-5 • ,
  • HUMIL6 Human Interleukin-6 Receptor Gene
  • HUMIL6B Interleukin-6 Gene
  • HBV Hepatitis B Virus
  • the illustrative nucleotides are: a. for HPV-11 beginning at nucleotide 927 201 5'-MTMCTTCTMCTMC-3 • , 202 5*-ZTMCTTCTMCTMC-3 • , b. for HPV-ll beginning at nucleotide 7101
  • the illustrative nucleotides are: a. for RSV beginning at nucleotide 1307
  • -MTMMMMMM-X 3 -CTTCTTM-5 » , -MTMMMM-X 3 -CTTCTTZ-5' , -ZTMMMMMM-X 3 -CTTCTTZ-5 ' , -ZTMMMMMM-X 3 -CTTCTTM-5• , -MTMMMMMC-X 3 -MTTMTTM-5 • , -MTMMMMMC-X 3 -MTTMTTZ-5 • , -ZTMMMMMC-X 3 -MTTMTTZ-5 l , -ZTMMMMMC-X 3 -MTTMTTM-5 • .
  • the illustrative nucleotides are: a. for HSV beginning at nucleotide 52916 701 5'-MMMTTTMCTTTMTMCTTT-3 ' ,
  • Cytomegalovirus (CMV) , the illustrative nucleotides are: a. for CMV beginning at nucleotide 176

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Agents qui se lient au sillon principal de duplexes d'acide nucléique de manière spécifique à certaines séquences et sont capables de former des liaisons covalentes avec un ou les deux brins du duplex en l'absence de lumière et sont utiles en tant qu'agents thérapeutiques dans le traitement d'états pathologiques dus à l'ADN duplex. Ces agents sont conçus de manière à ce que la réactivité de l'agent de réticulation n'interfère pas avec la spécificité de la séquence de l'agent qui se lie au sillon principal. Ainsi, des duplexes d'ADN spécifiques désirés peuvent-ils être ciblés et leur activité diminuée ou augmentée.
EP19910911335 1990-05-25 1991-05-24 Sequence-specific nonphotoactivated crosslinking agents which bind to the major groove of duplex dna Withdrawn EP0531436A4 (en)

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US52934690A 1990-05-25 1990-05-25
US529346 1990-05-25
US64065491A 1991-01-14 1991-01-14
US640654 1991-01-14

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EP0531436A1 true EP0531436A1 (fr) 1993-03-17
EP0531436A4 EP0531436A4 (en) 1993-06-16

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EP (1) EP0531436A4 (fr)
JP (1) JPH06502388A (fr)
AU (1) AU7998491A (fr)
CA (1) CA2083719A1 (fr)
WO (1) WO1991018997A1 (fr)

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US5849482A (en) * 1988-09-28 1998-12-15 Epoch Pharmaceuticals, Inc. Crosslinking oligonucleotides
US5824796A (en) * 1988-09-28 1998-10-20 Epoch Pharmaceuticals, Inc. Cross-linking oligonucleotides
USRE38416E1 (en) 1988-09-28 2004-02-03 Epoch Biosciences, Inc. Cross-linking oligonucleotides
AU9094991A (en) * 1990-11-23 1992-06-25 Gilead Sciences, Inc. Triplex-forming oligomers containing modified bases
US6136601A (en) * 1991-08-21 2000-10-24 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
AU3250093A (en) 1991-12-12 1993-07-19 Gilead Sciences, Inc. Nuclease stable and binding competent oligomers and methods for their use
WO1993012230A1 (fr) * 1991-12-13 1993-06-24 Sri International Formation d'une chaine helicoidale triple aux faisceaux (punpyn).(punpyn)
WO1993018187A1 (fr) * 1992-03-13 1993-09-16 California Institute Of Technology Reconnaissance de la triple helice de l'adn
AU6296294A (en) 1993-01-26 1994-08-15 Microprobe Corporation Bifunctional crosslinking oligonucleotides adapted for linking to a desired gene sequence of invading organism or cell
EP0830368A1 (fr) 1995-06-07 1998-03-25 Genta Incorporated Nouveaux lipides cationiques a base de carbamate
US6127533A (en) 1997-02-14 2000-10-03 Isis Pharmaceuticals, Inc. 2'-O-aminooxy-modified oligonucleotides
US6172209B1 (en) 1997-02-14 2001-01-09 Isis Pharmaceuticals Inc. Aminooxy-modified oligonucleotides and methods for making same
US6576752B1 (en) 1997-02-14 2003-06-10 Isis Pharmaceuticals, Inc. Aminooxy functionalized oligomers
US6673912B1 (en) 1998-08-07 2004-01-06 Isis Pharmaceuticals, Inc. 2′-O-aminoethyloxyethyl-modified oligonucleotides
US6043352A (en) 1998-08-07 2000-03-28 Isis Pharmaceuticals, Inc. 2'-O-Dimethylaminoethyloxyethyl-modified oligonucleotides
DE19840044A1 (de) * 1998-09-02 2000-03-23 Biotecon Ges Fuer Biotechnologische Entwicklung & Consulting Mbh Oligonukleotide, Verfahren und Kit zur Detektion von Listeria monocytogenes durch Nukleinsäureamplifikation und/oder -hybridisierung
US7125945B2 (en) 2003-09-19 2006-10-24 Varian, Inc. Functionalized polymer for oligonucleotide purification

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EP0375408A1 (fr) * 1988-12-20 1990-06-27 Baylor College Of Medicine Méthode de préparation d'oligonucléotides synthétiques se liant spécifiquement à des cibles sur des molécules d'ADN bicaténaire en formant un complexe tricaténaire colinéaire, les oligonucléotides synthétiques et méthodes d'utilisation
WO1990015884A1 (fr) * 1989-06-19 1990-12-27 The Johns Hopkins University Formation de complexes a triple helice d'adn a double brin par utilisation d'oligomeres de nucleosides
US5112962A (en) * 1989-04-19 1992-05-12 Northwestern University Labile anchors for solid phase polynucleotide synthesis

Patent Citations (3)

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EP0375408A1 (fr) * 1988-12-20 1990-06-27 Baylor College Of Medicine Méthode de préparation d'oligonucléotides synthétiques se liant spécifiquement à des cibles sur des molécules d'ADN bicaténaire en formant un complexe tricaténaire colinéaire, les oligonucléotides synthétiques et méthodes d'utilisation
US5112962A (en) * 1989-04-19 1992-05-12 Northwestern University Labile anchors for solid phase polynucleotide synthesis
WO1990015884A1 (fr) * 1989-06-19 1990-12-27 The Johns Hopkins University Formation de complexes a triple helice d'adn a double brin par utilisation d'oligomeres de nucleosides

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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY vol. 113, 25 September 1991, WASHINGTON, DC US pages 7765 - 7766 J.-P. SHAW ET AL. *
NUCLEIC ACIDS RESEARCH. vol. 19, no. 7, 11 April 1991, ARLINGTON, VIRGINIA US pages 1527 - 1532 R.H. ALUL ET AL. *
See also references of WO9118997A1 *

Also Published As

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CA2083719A1 (fr) 1991-11-26
WO1991018997A1 (fr) 1991-12-12
JPH06502388A (ja) 1994-03-17
AU7998491A (en) 1991-12-31
EP0531436A4 (en) 1993-06-16

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